Plant-based production of recombinant proteins has emerged as an efficient and cost-effective alternative to microbial fermentation and mammalian cell culture systems. Chloroplasts harbor high plasmid copy numbers and can be stably transformed, making them efficient platforms for protein production. In the present study, we used green fluorescent protein (GFP) as a reporter to compare the three major chloroplast promoters (rrn, psbA, and rbcL) involved in protein production in Nicotiana tabacum cv. “Petit Havana.” Three chloroplast transformation vectors were constructed, each regulated by a different promoter, and the transformation was performed via biolistic particle bombardment. Transformants were selected based on spectinomycin resistance and were confirmed by PCR. Among the three promoters, psbA showed the highest transformation efficiency and protein expression levels. Reverse transcription quantitative PCR showed that the mRNA levels (relative to Actin) for psbA (218.21±19.64) were nearly twice that of rbcL (126.60±8.78), and five times that of rrn (43.27±1.57). This transcriptional hierarchy was also observed at protein level. Immunoblotting showed the GFP levels (relative to psbA) were: psbA (1.00), rbcL (0.87), and rrn (0.77), whereas quantification through ELISA revealed relative GFP concentrations of: 616.2±28.7 ng/g LFW for psbA, 510.3±32.4 ng/g LFW for rbcL, and 338.9±100.2 ng/g ng/g LFW for rrn. These quantitative results demonstrate the importance of promoter selection for efficient expression of recombinant proteins in chloroplasts and show that the psbA promoter is suitable for high-efficiency chloroplast expression systems, providing a foundation for advancing plant-based molecular farming.

Wheat (Triticum aestivum L.) is one of the world's three staple crops and accounts for approximately 20% of the total calories consumed by the world's population. It is known that wheat is a difficult crop to introduce foreign genes into, having a large genome (16 Gb) containing three highly related subgenomes (AABBDD). Owing to the low transformation efficiency of wheat, it is difficult to apply new technologies such as genome editing and basic research based on molecular biology, such as the discovery of useful genes and functional analysis. Recently, the completion of a wheat genome map by the International Wheat Genome Sequencing Consortium (IWGSC) and the development of a stable and reproducible wheat transformation system have accelerated research regarding the expression and control of useful genes. In this review, we introduce in detail the recently developed highly efficient Agrobacterium-mediated wheat transformation system and its applications in plant biotechnology, such as RNA interference (RNAi), overexpression, and gene editing using this system.

AbstractThe SHORT VEGETATIVE PHASE (SVP) gene encodes a MADS-box gene family of transcription factors that repress floral transition. To explore the function of the Brassica rapa SVP (BrSVP) gene during the flowering time of this species, a construct containing BrSVP under the control of the cauliflower mosaic virus 35S promoter was introduced into B. rapa via Agrobacterium-mediated transformation. The resulting transgenic plants showed delayed flowering time, and RT-PCR analyses further revealed that BrSVP repressed the expression of the floral integrator genes AGL20, AGL24, and FT during vernalization. Our data indicated that BrSVP acts as a negative regulator in the flowering time of B. rapa and that it may therefore be a useful genetic source for crop improvement with respect to flowering time regulation.

Maize is the most important grain crop in the world. Genetic engineering technology has been used to enhance its various agronomical traits. The transformation of maize is a crucial step in the application of gene technologies to improve maize. The choice of genotype and explant material influences the transformation efficiency and the production of stable transgenic plants. Immature embryos of Hi IIA were infected with Agrobacterium tumefaciens LBA4404 including superbinary vectors (bar and GUS or GFP genes). The transformation efficiency was based on transgenic calli induction from immature embryos on the selection medium with 3 mg/L bialaphos. The transformation efficiency varied from 1.01 to 2.74%. The integration and expression of bar, GUS, and GFP genes were confirmed in T₀ and T₁ generations of transgenic plants using genomic PCR and the bar strip test. In addition, herbicide resistance in T₁ transgenic plants was observed when leaves and whole plants were treated with Basta. These results suggest that the successful Agrobacterium-mediated transformation of Hi IIA will improve further opportunities for functional genomic and genome editing studies in maize.

Plant molecular farming has attracted a lot of attention lately in the field of mass production of industrially valuable materials by extending application of the plant as a kind of factory concept. Among them, protein expression system using rice(Oryza sativa L.) callus is a technology capable of mass culture and industrialization because of a high expression rate of a target protein. This study was carried out to develop an Agrobacterium-mediated transformation system to increase the utilization of rice callus. The transformation efficiency was improved by using the hand when seeds were de-husked for callus induction. Furthermore, we were possible induction of callus from 6 years old seed smoothly. Selection of the callus contained the target gene was required a cultivation period of at least 3 weeks, and the most efficient selection period was after 6 weeks of culture including one passage. This selection was confirmed that the gene was stably inserted into the genomic DNA of the plant cell by the southern blot analysis and progeny test. Such an efficient selection system of rice callus that can be cultured in the long term will be contribute to the industrialization of useful recombinant proteins using rice.

This study was conducted to isolate a salt tolerant gene and to develop salt tolerant rice for reclaimed-saline areas through genetic transformation. A rice c/DRE binding factor 4 (OsCBF4) cDNA was isolated from rice (cv. Nipponbare) using RT-PCR. The full-length cDNA of the CBF4 gene consists of 1,429 nucleotides and 274 amino acid residues. The OsCBF4 shares from 33 to 49% identity of deduced amino acid sequence with other CBFs of rice. In order to develop salt tolerant rice, transgenic rice plants containing the OsCBF4 gene were obtained via Agrobacterium-mediated transformation. The stable incorporation of the OsCBF4 gene into rice genome was confirmed by PCR and Southern analysis. The stable expression of introduced gene was also validated by RT-PCR analysis in T₂ plants. Biological assay of T₃ progeny of the transgenic plants in Yoshida solution containing 120 mM Nacl for 2 weeks, confirmed that the OsCBF4 confers salt tolerance to transgenic rice plants. OsCBF4 transgene in the transgenic line CBF4-10 was markedly expressed up to over three-fold in the leaf by 120 mM NaCl treatment. Real-time PCR analysis revealed that the levels of the transgene expression were markedly increased under salt treatment. The transgenic line CBF4-10 which showed highest ability to recover from the saline stress could be used as a potential source for salt tolerance in rice breeding programs.